The MRDS is currently involved in two major projects. One project is investigating the molecular mechanism(s) of 7-ketocholesterol (7KCh)-mediated toxicity and inflammation. The other project involves the development of 7KCh-mediated angiogenesis models in rat and rabbit for the purpose of testing drugs that may be useful in the treatment of age-related diseases where chronic inflammation resulting from the accumulation of oxidized lipids may be involved. 7-KCh is an oxidized derivative of cholesterol found in very high concentrations in atherosclerotic plaques (as much as 25% weight) and in the back of the retina in deposits in Bruchs membrane (BM) and the choriocapillaris (CH). 7-KCh forms by the autooxidation of cholesterol and especially cholesterol-fatty acid esters (CEs) in the presence of oxygen and a transition metal such as Cu+2 or Fe+2. This is the main reason why the CE-rich LDL deposits are so readily oxidized. A recent and yet unpublished study using monkey eyes has shown a large increase in 7KCh in the RPE and CH with aging. Monkey 16 years of age and older contain 60-70 times more 7KCh than monkeys less than 5 years of age. Drusen deposits also seem to contain large amounts of 7KCh. Preliminary results using frozen sections of drusen-containing human eyes indicate that the drusen-containing sections have 7KCh levels 100 times greater than sections without drusen. A more comprehensive collaborative study is in progress to determine the levels of 7KCh in drusen deposits. We have also added several dozen new monkey analyses from the neural retina and RPE/choriocapillaris to our previous study. The results clearly demonstrate that the older the animal the greater the levels of 7KCh in the RPE/choroid tissues. The precise mechanism of action of 7KCh is not known. Studies performed by the MRDS and by other investigators have shown that 7-KCh causes endoplasmic reticulum (ER) stress and sets off a variety of inflammatory pathways mostly mediated by NFkB. Using sterculic acid (a potent 7KCh antagonist) and a variety of other inhibitors and siRNAs we have been able to systematically eliminate all of the inflammatory pathways downstream from the ER. Presently the MRDS is focusing on determining which of the upstream pathways that lead to ER stress are activated by 7KCh and inhibited by sterculic acid. Recent evidence suggests that the TLR-4 receptor is significantly involved but not the sole source of the inflammatory response. Complete inhibition of NFkB can attenuate but not ablate the inflammatory response. This suggests that 7KCh can also activate of multiple inflammatory pathways that are unrelated to ER stress. The metabolism of 7KCh outside of the liver is not known. Various investigators have reported hydroxylation by the cytochrome P450, CYP27A or sulfation by SULT2B1. These enzymes are able to use 7KCh as substrate in vitro but not in vivo. SULT2B1 expression in the retina is nil and CYP27A1 doesn't seem to come in contact with 7KCh. 7-KCh localized to lipid deposits in Bruchs membrane and in the capillary endothelial cells of the choriocapillaris and the neural retina. We have extensively searched using LCMS for hydroxylated and sulfated 7KCh metabolites in both cultured cells treated with 7KCh and in retinal and RPE extracts from monkeys and humans and have not found any. However, what we do find is an abundance of 7KCh-fatty acids esters (7KFAEs) in both in vitro and in vivo extracts. Our experiments indicate that these esters are formed by ACAT-1 (SOAT-1) in conjunction with the calcium-dependent PLA2. In vitro we have found that as much as 25% of the 7KCh that enters the cell is converted into 7KFAEs. We have also found that HDL is crucial in promoting 7KCh efflux and this occurs independently of the ABCA1 and ABCG1 transporters. Commercially purchased HDL also contains significant amounts of 7KFAEs. This suggests that HDL is a major transporter of 7KCh and its esters back to the liver. The MRDS has also been involved in developing an angiogenesis model in rats and rabbits. In this model a 0.5 mm disc- shape implant containing 7KCh (10% w/w) mixed with hydrogel and polyethylene glycol is placed in the anterior chamber of the rat eye. Within 7 days the 7KCh-containing implant becomes tumor-like doubling in size and drawing large numbers of neovessels from the limbus. Implants containing cholesterol only dissolve and dissipate without causing any inflammatory response and angiogenesis. This not only demonstrates the ability of 7KCh to cause angiogenesis but also has created a very useful angiogenesis model. The transparency of the cornea allows the monitoring and quantification of the neovessels by fluorescein angiography using a dissecting microscope equipped with a fluorescent lamp and a camera. Anti-angiogenic drugs are being tested by simply incorporating them directly into the implants or by eye-drop delivery. Aside from sterculic acid and sterculia oil we have found two naturally occurring novel compounds that inhibit the 7KCh-mediated inflammatory response. One compound is present in the AMLA fruit extract (Embilica Officianalis, or Indian gooseberry) and another in the sterculia foetida leaves. The chemical structure of these compounds is under investigation. In summary, the MRDS has made considerable progress in defining the mechanism of action of 7KCh, its metabolism and its potential role in age-related diseases. Is has also developed a very useful rat angiogenesis model for drug testing. Using this model the MRDS has demonstrated that sterculic acid and sterculia oil may be useful in the treatment of angiogenesis associated with oxidized lipids. The new compounds found may be significantly more potent than sterculic acid. Once the chemical structures are determined this will provide additional insight for future small molecule development for the treatment of chronic inflammation and angiogenesis.
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